The molecular regulation of phenotypic establishment was studied during neuronal differentiation through the identification of embryonic-specific trans-factors and their corresponding cis-elements, the role of gene structure, chromatinization and methylation. The rat and mouse enkephalin and VIP genes were isolated, characterized, and used as models of phenotypic genes. Nuclear extracts of different brain regions and peripheral tissues from animals of various ages, developing primary neuronal cell cultures, tumor cell lines and transgene animals were employed as model systems as well. Experiments with cycloheximide showed that phenotypic establishment (embryonic expression of the phenotype) is critically dependent on de novo protein biosynthesis. Mobility shift assays with different fragments of the enkephalin gene cis-elements showed numerous specific complexes exclusively present in certain, but not all, regions of the prenatal brain and absent from adult brain as well as from peripheral tissues (adrenal, liver).We found elaborate, tissue-, cell-and age-dependent binding patterns indicating the existence of large number of embryonic- and cell-specific DNA- binding proteins. Preincubation of nuclear extracts with embryonic- specific monoclonal antibodies recognized several DNA-binding proteins and resulting "supershifts". A Ca2+ -induced "supershift" specific to the fragment of the rat ppENK gene that contains the sequence motif (TG/CA)56 between -614 and -670 positions (presumed Z-DNA) was observed. Gel shift assays with ds(TG)28/(AC)28 (Z-DNA) probe at low Ca2+/Mg2+ concentration revealed the presence of specific binding proteins in nuclear extracts of peripheral tissues (liver, adrenal) which were absent in CNS. Using a variety of techniques it was demonstrated that: a) only the presumed Z-DNA sequence motif (TG/CA)n, but no other random sequences tested, was capable of "DNA-slippage" or sliding, in a Ca2+/Mg2+-dependent manner; b) the sliding was concentration dependent and maximal at the concentration of 1-3 mM; c) this "slippage/sliding" did not require the presence of protein at all; d) the direction of the sliding was strictly unidirectional (3' to 5'). The mouse VIP chromosomal gene was extensively characterized and sequenced and showed the presence of the presumed Z-DNA sequence motif (TG/CA)38 and high homology to the human gene. Immunohistochemical studies on developing DRG neurons showed a temporary, developmental-specific intranuclear localization of CGRP, the neuropeptide involved in noceffector function. The activity-dependent regulation of the synaptic specific phosphoprotein GAP-43 was studied in primary DRG neurons and no activity-dependent regulation was found at the transcriptional level.